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Troubleshooting table.

Step Problem Possible reason Solution

Choose appropriate membranes with


Adsorption of protein on
14 Loss of protein low binding capacity (polyethersulfone
to membrane surfaces
membrane are often appropriated)

Nonspecific aggregation Conformational changes


15 during concentration due to variations of the Select an appropriate protein buffer pH
process environment

Protein denaturation. The


tendency of the protein Foaming and air bubbles Minimize shaking and use special care
20 solution to foam upon formation may create an on handling the macromolecular
gentle stirring can be a oxidizing environment solution
sign of denaturation

Crystals can sometimes


adhere to the drop holder
surface making the Heterogeneous
removal difficult. This nucleation and growth
Use a crystal probe to remove the
28 disadvantage can turn that produce crystals
crystal
into an advantage where sticking tenaciously to
occasionally the surface the support
of the drop holder can
assist the nucleation

Depends on the physical Set up screening experiments with


Protein characterized by properties of the drops containing protein
low solubility protein. Whatever solution/precipitant solution ratio
(concentration lower than crystallization method is greater than 1:1. The increase of
28 3 mg ml-1). Low protein used, it requires high protein amount and drop size may
concentration may concentrations of produce larger crystals. Bigger drops
produce very small protein as compared to can give bigger crystals, because there
crystals normal biochemical is more protein in the drops and the
operations rate of equilibration is slower

Consider diagnostic testing (i.e., SDS–


The protein sample is polyacrylamide gel electrophoresis,
heterogeneous and mass spectroscopy, isoelectric
impure, not only in focusing, activity, light scattering,
Most of the drops have a
terms of contaminating circular dichroism and nuclear
35 heavy amorphous
agents, but also in magnetic resonance (NMR) as in
precipitate
terms of sequence PRELIMINARY SAMPLE PREPARATION
integrity and AND CHARACTERIZATION) to measure
conformation the homogeneity and purity of the
protein

54 The optimization process It is a nonlinear process Carefully plan additional trials with
is often more complex and one variable may be matrices encompassing a parameter
and difficult than one coupled to another range spanning the conditions that
might expect have provided the 'hit'. Vary pairs of
Step Problem Possible reason Solution

the above parameters or factors


following the order reported in Step 55

Too much of your final


wash solution added to
Remove excess liquid with a small thin
the sitting drop;
strip of filter paper or a very thin
concentration of
84 Dissolving seeds capillary. Lightly increase the
precipitant too low in
precipitant concentration in final wash.
final wash; precipitant
Increase the precipitant concentration
concentration too low in
either drop or reservoir

Improperly washed seed


that generates
Increase the series of washes, slightly
microseeds or unwanted
reduce precipitant concentration and
Too many nucleation nuclei in the drop being
reduce equilibration rate by a decrease
sites seeded, precipitant
in reservoir/drop volume ratio (see
concentration too high,
Step 66)
rate of equilibration too
high

Microseed solution has


been stored at 4 °C for Use only freshly prepared microseed
too long. Microseed solution6. Operate with more diluted
Microseeding does not
solution contains too microseed solution till 10-7 and/or
89 work, too many
many nuclei and/or two according the above Step 84 solutions;
nucleation sites
last points in Step 84 seed drops at variable protein
above; protein drop concentration
concentration is too high

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